A relation between inhibition of protein synthesis and conformation of 5'-phosphorylated 7-methylguanosine derivatives.
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Animals↗
Biomedical subjects
L A Weber
Publications and source records attributed to L A Weber.
Histone mRNAs contain blocked and methylated 5' terminal sequences but lack methylated nucleosides at internal positions.
Histone mRNA, labeled with 32P or 3H-methionine during the S phase of partially synchronized HeLa cells, was isolated from the polyribosomes and purified as a "9S" component by sucrose gradient sedimentation. We identified two types of 5' terminals, m7G(5')pppNmpN and m7G(5')pppNm-pNmpN, in which the first methylated nucleoside is 7-methylguanosine, the second is either N6, 2'-O-dimethyladenosine, 2'-O-methyladenosine, or 2'-O-methylguanosine, and the third is 2'-O-methyluridine, 2'-O-methylcytidine, or 2'-O-methyladenosine. Approximately 1.7% of the 32P label was present in the 5' terminal structures. Assuming a similar specific radioactivity for all phosphates, this percentage corresponds to an average of one terminal per 335 nucleotides. Histone mRNA differed from bulk polyadenylylated mRNA of HeLa cells in lacking significant amounts of 2'-O-methyluridine or 2'-O-methylcytidine in the second position of the 5' terminal oligonucleotide and in lacking N6-methyladenosine residues at internal positions.
Adenosine↗
5'-Terminal 7-methylguanosine and mRNA function: influence of potassium concentration on translation in vitro.
Vaccinia mRNAs containing either 5'-terminal m7G or unmethylated 5'-terminal structures were synthesized in vitro and their relative efficiencies of translation were compared in wheat germ and reticulocyte cell-free protein-synthesizing systems. The importance of the m7G group for efficient translation increases as the K+ concentration is raised. At K+ concentrations optimal for translation of mRNA containing m7G, unmethylated mRNA is translated at the same relative low efficiency in both cell extracts. The rate of binding of mRNA to ribosomes at K+ concentrations close to those found in intact cells is strongly influenced by the presence of m7G regardless of the source of the cell extract.
Animals↗
Inhibition of HeLa cell messenger RNA translation by 7-methylguanosine 5'-monophosphate.
Translation of HeLa cell RNA containing poly(A) in a wheat germ cell-free system is markedly but incompletely inhibited by 7-methylguanosine 5'-monophosphate (m7G5'p). We have analyzed the translation products synthesized in the presence of different concentrations of m7G5'p and find that translation of all mRNAs is equally inhibited. To demonstrate the specificity of the inhibitor for RNAs with 5'-terminal m7G5' ppp... we show that specific translation products of satellite tobacco necrosis virus RNA, which does not have this 5' terminus, are synthesized in the presence of m7G5' p. Protein synthesis programmed by endogenous mRNA in a HeLa cell-free system is inhibited after a 10-min lag by m7G5' p. Other guanosine nucleotides without the 7-methyl group or with the phosphate in a different position are not inhibitor. We show that translation of all mRNAs is inhibited to a similar extent by m7G5'p in the HeLa cell-free system, by synthesizing 35S-labeled proteins in the presence of different inhibitory concentrations of this nucleotide and analyzing the translation products by electrophoresis and autoradiography. Translation of encephalomyocarditis virus RNA added to the HeLa cell-free system is not inhibited by m7"g5p; this viral RNA does not have this nucleotide at the 5' terminus. This indicates that m7G5'p specifically inhibits translation of mRNAs with the 5' terminus m7G5'ppp... and suggests that initiation of translation of picornavirus RNA may proceed via a mechanism different from that of cellular mRNAs.
Globins↗
Evidence for role of m7G5'-phosphate group in recognition of eukaryotic mRNA by initiation factor IF-M3.
7-methylguanosine 5'-monophosphate inhibits protein synthesis in a fractionated, messenger-dependent, reticulocyte cell-free system. This compound also inhibits binding of histone mRNA to reticulocyte ribosomes as well as interaction of VSV mRNA and histone mRNA but not EMC virus RNA with purified initiation factor IF-M3. These studies provide evidence that the role of 7-methylguanosine in the mechanism for initiation of eukaryotic mRNA translation may be related to specific recognition of mRNA by initiation factor IF-M3.
Animals↗
Translation of RNA from unfertilised sea urchin eggs does not require methylation and is inhibited by 7-methylguanosine-5'-monophosphate.
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Animals↗
Inhibition of initiation of protein synthesis by 7-methylguanosine-5'-monophosphate.
Translation of rabbit globin mRNA in a wheat germ protein-synthesizing system is inhibited by the nucleotide 7-methylguanosine-5'-monophosphate (m7G5'p) but not by other guanosine nucleotides without the 7-methyl group or with the phosphate in a different position. Translation of RNA of tobacco mosaic virus and poly(A) + HeLa RNA is also inhibited by m7G5'p. We show that m7G5'p prevents the association of mRNA with ribosomal subunits to form an initiation complex. We propose that m7G5'p interacts with a site on initiation factor(s) or ribosomes which is involved in mRNA recognition, presumably by binding to the 5'-terminal sequence m7G5'ppp. m7G5'p does not inhibit translation of poly(U) and RNA of satellite tobacco necrosis virus, which do not have the 5'-terminal sequence m7G5'ppp. In the case of RNA of satellite tobacco necrosis virus, some stimulation of its translation is consistently observed in the presence of m7G5'p; possible interpretations of this finding are discussed.
Cell-Free System↗
A cell free system from HeLa cells active in initiation of protein synthesis.
A cell free system programmed by endogenous mRNA and active in initiation of protein synthesis has been obtained from HeLa cells by adding 25-100 muM hemin to the medium used to homogenize the cells. Hemin stabilizes the initiation activity of the extract, which otherwise decays rapidly even at 0 degrees C. The role of hemin in promoting initiation has been examined by fractionating the extracts into ribosomes and postribosomal supernatant (S150). An extract prepared without hemin or the S150 obtained from this extract prepared without hemin or the S150 obtained from this extract inhibits protein synthesis of the extract containing hemin by about 30%. The ribosomes prepared from extracts containing hemin are active in initiation of protein synthesis, whereas the ribosomes obtained from the extracts prepared without hemin show little or no initiation. These results have suggested that addition of hemin prevents the formation of an inhibitor of initiation in the S150 and at the same time protects from inactivation an initiation factor associated with ribosomes or ribosomal subunits. Addition of 2 mM GTP to HeLa extracts stabilizes the initiation activity, though to a smaller degree than hemin. The effects of hemin and GTP are not additive, suggesting that they may act on the same target molecule, though possibly by different mechanisms. The mechanism of action of GTP is discussed in view of similar observations made in the rabbit reticulocyte cell free system.
Cell-Free System↗